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  • Casein Kinase I (CK1)

    Description

    Casein Kinase I (CK1) is a serine/threonine protein kinase (1). It is a truncated monomer (1-317) of the CK1d isoform, which lacks the regulatory C-terminal domain, containing 111 amino acids (2). In vitro studies have shown that the activity of CK1δ is regulated by autophosphorylation of its C-terminal domain. Autophosphorylation of this domain on potential sites leads to inhibition of kinase activity (3). There are at least seven mammalian CK1 isoforms and their splice variants, and distinct CK1 family members have a variety of roles in eukaryotic cells (4). 

    Highlights

    • Protein serine/threonine kinase
    • Rat, recombinant (E. coli)

    Product Source

    Isolated from a strain of E. coli that carries a clone expressing CK1 δ derived from a rat testis cDNA library (kindly provided by Dr. P. J. Roach). Two codons, Ser-318 and Arg-319, have been changed to stop codons, resulting in a truncation of the C-terminal portion of the expressed protein (2).

    Recognition Determinant

    The most effective recognition motif for phosphorylation by CK1 is pSXXS/T where Ser in the position -3 is phosphorylated (3). Also, the clusters of 3 or 4 acidic residues ending at the position -3, preferably Asp, can specify phosphorylation by CK1. However, the substrates so formed are much poorer than those containing phosphate groups (5).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CK1 Reaction Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of CK1 required to catalyze the transfer of 1 pmol of phosphate to CK1 Phosphopeptide Substrate, KRRRALpSVASLPGL (70 µM), in 1 minute at 30°C in a total reaction volume of 25 µl.

    Reaction Conditions

    1X CK1 Reaction Buffer
    Supplement with 200 μM ATP
    Incubate at 30°C

    1X CK1 Reaction Buffer:
    50 mM Tris-HCl
    10 mM MgCl2
    5 mM DTT
    pH 7.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    2 mM DTT
    1 mM Na2EDTA
    1 mM EGTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.0 @ 25°C

    Molecular Weight

    Theoretical: 36 kDa

    Specific Activity

    2,000,000 units/mg

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    Notes

    1. Reaction Conditions: 1X CK1 Reaction Buffer, supplement with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.
    2. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    3. If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM. However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate. To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed.
      Recommended reference: 
      Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
    4. If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.

    References

    1. Hathaway, G.M. and Traugh, J. A. (1979). J. Biol. Chem. 254, 762-768.
    2. Graves, P.R., Haas, D.W., Hagedorn, C.H., DePaoli-Roach, A.A. and Roach, P.J. (1993). J. Biol. Chem. 268, 6394-6401.
    3. Graves, P.R., and Roach, P.J. (1995). J. Biol. Chem. 270, 21689-21694.
    4. Knippschild, U. et al. (2005). Onkologie. 28, 508-514.
    5. Iotow, H. and Poach, P.J. (1991). J. Biol. Chem. 266, 3724-3727.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. How much Casein Kinase I (NEB# P6030) should be used?
    2. What is the consensus sequence for CKI (P6030)?

    Selection Tools

    Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
    If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 µM.
    1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
    The most effective recognition motif for phosphorylation by CK1 is pSXXS/T where Ser in the position -3 is phosphorylated